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US6872850B2 - Small molecule inhibitors of secretion of proteins encoded by ARE-mRNAs - Google Patents

Small molecule inhibitors of secretion of proteins encoded by ARE-mRNAs Download PDF

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US6872850B2
US6872850B2 US10/117,955 US11795502A US6872850B2 US 6872850 B2 US6872850 B2 US 6872850B2 US 11795502 A US11795502 A US 11795502A US 6872850 B2 US6872850 B2 US 6872850B2
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mes
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phenyl
secretion
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US20030199453A1 (en
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Tony Giordano
Michael A. Sturgess
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Message Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
    • C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
    • C07C281/18—Compounds containing any of the groups, e.g. aminoguanidine the other nitrogen atom being further doubly-bound to a carbon atom, e.g. guanylhydrazones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/56—Amides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/02—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines
    • C07D217/06—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with only hydrogen atoms or radicals containing only carbon and hydrogen atoms, directly attached to carbon atoms of the nitrogen-containing ring; Alkylene-bis-isoquinolines with the ring nitrogen atom acylated by carboxylic or carbonic acids, or with sulfur or nitrogen analogues thereof, e.g. carbamates

Definitions

  • the invention relates to methods and compounds that decrease secretion of proteins encoded by ARE-mRNAs, such as Tumor Necrosis Factor (TNF ⁇ ), or that modulate regulation of ARE-mRNAs.
  • ARE-mRNAs such as Tumor Necrosis Factor (TNF ⁇ )
  • TNF ⁇ Tumor Necrosis Factor
  • ARE-mRNAs are mRNA molecules that contain an adenylate uridylate (AU)-rich element. AREs are usually found in the 3′ untranslated region (3′ UTR) of mRNAs rather than in the coding region. ARE regulatory regions modulate the rate of mRNA degradation and the rate of translation. A database of human mRNAs containing an ARE consensus sequence was recently reported (Bakheet et al., Nucleic Acids Research 29:246-254, 2001).
  • TNF ⁇ is a pleiotropic cytokine produced predominantly by activated monocytes/macrophages.
  • TNF ⁇ is a primary mediator of numerous immune functions, including hemorrhagic cytotoxicity, inflammation, and regulation of antiviral and immune proliferative and activation responses.
  • TNF ⁇ exhibits a potent cytolytic and cytostatic activity towards a variety of cells, including tumor cell lines and virally infected cells.
  • TNF ⁇ is a potent mediator of monocyte/macrophage migration, activation, and differentiation.
  • TNF ⁇ stimulates activated T-cells and B-cells and enhances neutrophil chemotaxis and phagocytic activity.
  • Endothelial cells are also primary targets of TNF ⁇ activity; for example, TNF ⁇ induces the release of IL-1 from endothelial cells and promotes endothelial cell procoagulant activity and neutrophil adhesion, which are involved in local inflammatory responses.
  • TNF ⁇ also stimulates fibroblast migration, proliferation, and cytokine production.
  • the present invention features compounds and methods for inhibiting secretion of proteins encoded by ARE-mRNAs, such as Tumor Necrosis Factor (TNF ⁇ ), or modulating regulation of ARE-mRNAs.
  • the compounds of the present invention may be used as therapeutics to inhibit TNF ⁇ secretion in a desired cell, such as a cell of the immune system (e.g., a macrophage, monocyte, T-cell, B-cell, granulocyte, eosinophil, mast cell, fibroblast, smooth muscle cell, chondrocyte, osteoblast, glial cell, keratinocyte, or endothelial cell).
  • a cell of the immune system e.g., a macrophage, monocyte, T-cell, B-cell, granulocyte, eosinophil, mast cell, fibroblast, smooth muscle cell, chondrocyte, osteoblast, glial cell, keratinocyte, or endothelial cell.
  • diseases including but not limited to
  • the invention features a compound having the formula: or a derivative thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 2 and R 5 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • X is oxygen or H 2 N(HN)C(H)NN—, and m is at least one.
  • m is between 1 and 15, 1 and 10, or 1 and 5, inclusive.
  • m is one or two.
  • R 1 or R 6 is a methyl group.
  • R 2 or R 5 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 3 and R 4 are hydrogen.
  • R 2 is a phenyl group and R 5 is an isopropyl group, or R 2 is an isopropyl group and R 5 is a phenyl group.
  • the invention features a compound having the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 2 and R 5 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl.
  • M is at least one.
  • m is between 1 and 15, 1 and 10, or 1 and 5, inclusive. In other embodiments, m is one or two.
  • R 2 or R 6 is a methyl or phenyl group.
  • R 2 or R 5 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 3 and R 4 are hydrogen.
  • R 2 is a phenyl group and R 5 is an isopropyl group, or R 2 is an isopropyl group and R 5 is a phenyl group.
  • the invention provides a compound having the formula: or a derivative or salt thereof.
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group; R 3 and R 4 are independently selected from the group consisting of an alkyl and an aryl group.
  • X is oxygen or H 2 N(HN)C(H)NN—.
  • R 3 or R 4 is a phenyl or methyl group.
  • R 1 or R 2 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 1 is a phenyl group and R 2 is an isopropyl group, or R 1 is an isopropyl group and R 2 is a phenyl group.
  • the invention provides a compound having the formula: or a derivative or salt thereof.
  • R 1 and R 4 are independently selected from the group consisting of an alkyl or an aryl group.
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • R 1 or R 4 is a phenyl or methyl group.
  • R 2 or R 3 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 2 is a phenyl group and R 3 is an isopropyl group, or R 2 is an isopropyl group and R 3 is a phenyl group.
  • the invention features a compound having the formula: or a derivative or salt thereof.
  • R 1 and R 5 are independently selected from the group consisting an aryl or alkyl group;
  • R 2 is hydrogen, an alkyl, or aryl group.
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • X is oxygen or H 2 N(HN)C(H)NN—; and
  • m is at least one.
  • m is between 1 and 15, 1 and 10, or 1 and 5, inclusive.
  • m is one or two, and R 1 or R 5 is a methyl or phenyl group.
  • R 2 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 3 and R 4 are hydrogen.
  • the invention features a compound having the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 3 is hydrogen, an alkyl, or an aryl group.
  • R 2 , R 4 , and R 5 are independently selected from the group consisting of hydrogen and an alkyl.
  • M is at least one.
  • m is between 1 and 15, 1 and 10, or 1 and 5, inclusive.
  • m is one or two.
  • R 1 or R 6 is a methyl or phenyl group.
  • R 3 is a benzyl, 4-Cl-benzyl, 4-I-benzyl, 4-F-benzyl, 4-nitro-benzyl, 4-phenyl-benzyl, or 4-methoxy-benzyl group phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 2 , R 4 , and R 5 are hydrogen.
  • the invention provides a compound having the formula: or a derivative or salt thereof.
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • R 3 is an aryl group.
  • M is at least one.
  • m is between 1 and 15, 1 and 10, or 1 and 5, inclusive. In other embodiments, m is one or two.
  • R 1 or R 2 is a methyl, isopropyl, or phenyl group.
  • R 3 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 3 is an aryl group that has a substituent bound to carbon 4 of the aromatic ring.
  • the compounds of the present invention are useful for the prevention and treatment of a variety of conditions involving ARE-mRNAs or proteins encoded by ARE-mRNAs. Accordingly, the invention features a pharmaceutical composition that includes one or more of the compounds of the present invention in a pharmaceutically acceptable carrier.
  • a compound described in any of the embodiments of the above aspects of the invention may be used in any of the following clinical applications.
  • One such method for treating or preventing a condition in a subject involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 2 and R 5 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • X is oxygen or H 2 N(HN)C(H)NN—; and m is at least one.
  • the invention provides another method for treating or preventing a condition in a subject that involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 2 and R 5 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl.
  • M is at least one.
  • the invention provides yet another method for treating or preventing a condition in a subject that involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • R 3 and R 4 are independently selected from the group consisting of an alkyl and an aryl group.
  • X is oxygen or H 2 N(HN)C(H)NN—.
  • the invention provides still another method for treating or preventing a condition in a subject that involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 4 are independently selected from the group consisting of an alkyl and an aryl group; and R 2 and R 3 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • the invention provides another method for treating or preventing a condition in a subject that involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 5 are independently selected from the group consisting an aryl or alkyl group;
  • R 2 is hydrogen, an alkyl, or aryl group;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • X is oxygen or H 2 N(HN)C(H)NN—; and m is at least one.
  • the invention provides a method for treating or preventing a condition in a subject that involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 3 is hydrogen, an alkyl, and an aryl group.
  • R 2 , R 4 , and R 5 are independently selected from the group consisting of hydrogen and an alkyl, and m is at least one.
  • the invention provides another method for treating or preventing a condition in a subject that involves administering a compound to the subject in an amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA or modulate regulation of an ARE-mRNA in the subject.
  • the compound has the formula: or a derivative or salt thereof.
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • R 3 is an aryl group, and m is al least one. In various embodiments, R 3 is an aryl group that has a substituent bound to carbon 4 of the aromatic ring.
  • the invention features a method of forming a compound of interest by reacting
  • R 1 R 3 , R 6 and R 9 are each a protecting group.
  • R 2 and R 4 are independently selected from the group consisting of hydrogen and an alkyl.
  • R 5 , R 7 , R 8 , R 10 , and R 11 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • X is carbon, nitrogen, oxygen, or sulfur; and m and n are at least one. In various embodiments, m or n is between 1 and 15, 1 and 10, or 1 and 5, inclusive. In particular embodiments, m or n is one or two.
  • R 5 or R 8 is a benzyl, 4-Cl-benzyl, 4-I-benzyl, 4-F-benzyl, 4-nitro-benzyl, 4-phenyl-benzyl, 4-methoxy-benzyl, 3-acetyl-benzoyl, 4-benzyol-benzoyl, phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 2 , R 4 , or R 9 is hydrogen.
  • R 7 or R 10 is a methyl or phenyl group.
  • R 5 , R 7 , R 8 , R 10 , or R 11 includes, or is modified to include, a protecting group, such as a t-butyloxycarbonyl or benzyloxycarbonyl group.
  • a protecting group such as a t-butyloxycarbonyl or benzyloxycarbonyl group.
  • either one or two sixth compounds are incorporated into the compound of interest.
  • the method further includes reacting another sixth compound.
  • the method also includes reacting the second, third, fourth, or fifth compound with a condensing agent to accelerate its rate of reaction with another compound.
  • the method may also include removing a protecting group from an intermediate or from the compound of interest.
  • other third or fifth compounds that have additional substituents on the benzene ring may be used in this method.
  • a first compound having a substituent in place of one or both hydrogens of a methylene group (—CH 2 —) may be used.
  • the invention features a method of forming a compound of interest by reacting
  • R 1 , R 3 , and R 6 are each a protecting group.
  • R 2 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • R 5 , R 7 , R 8 , and R 9 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • X is carbon, nitrogen, oxygen, or sulfur; and m and n are at least one. In various embodiments, m is between 1 and 15, 1 and 10, or 1 and 5, inclusive. In particular embodiments, m is one or two.
  • R 5 is a benzyl, 4-Cl-benzyl, 4-I-benzyl, 4-F-benzyl, 4-nitro-benzyl, 4-phenyl-benzyl, 4-methoxy-benzyl, 3-acetyl-benzoyl, 4-benzyol-benzoyl, phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 2 or R 4 is hydrogen.
  • R 7 or R 8 is a methyl or phenyl group.
  • R 5 , R 7 , R 8 , or R 9 includes, or is modified to include, a protecting group, such as a t-butyloxycarbonyl or benzyloxycarbonyl group.
  • a protecting group such as a t-butyloxycarbonyl or benzyloxycarbonyl group.
  • either one or two fifth compounds are incorporated into the compound of interest.
  • the method further includes reacting another fifth compound.
  • the method also includes reacting the second, third, or fourth compound with a condensing agent to accelerate its rate of reaction with another compound. The method may also include removing a protecting group from an intermediate or from the compound of interest. If desired, other third or fourth compounds that have additional substituents on the benzene ring may be used in this method.
  • a first compound having a substituent in place of one or both hydrogens of a methylene group (—CH 2 —) may be used.
  • the invention features a method of forming a compound of interest by reacting
  • R 4 is hydrogen
  • R 1 , R 2 , R 3 , R 5 , and R 6 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • X is carbon or nitrogen
  • m is at least one. In various embodiments, m is between 1 and 15, 1 and 10, or 1 and 5, inclusive. In particular embodiments, m is one or two.
  • R 1 , R 2 , R 3 , or R 5 is a benzyl, 4-Cl-benzyl, 4-I-benzyl, 4-F-benzyl, 4-nitro-benzyl, 4-phenyl-benzyl, 4-methoxy-benzyl, 3-acetyl-benzoyl, 4-benzyol-benzoyl, phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 6 is an aryl group, such as a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 6 is an aryl group that has a substituent bound to carbon 4 of the aromatic ring.
  • R 1 , R 2 , R 3 , R 5 , or R 6 includes, or is modified to include, a protecting group, such as a t-butyloxycarbonyl or benzyloxycarbonyl group.
  • the method also includes reacting the first compound with a condensing agent to accelerate its rate of reaction with the second compound. The method may also include removing a protecting group from the compound of interest.
  • the alkyl group is a linear or branched saturated hydrocarbon group of 1 to 5, 1 to 10, 1 to 20, 1 to 50, or 1 to 100 carbon atoms; such as a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, or tetradcyl group; or a cycloalkyl group, such as a cyclopentyl or cyclohexyl group.
  • suitable alkyl groups include heteroalkyl groups in which one or more carbons have been replaced with another atom, such as nitrogen, sulfur, oxygen, or phosphate.
  • One or more of the hydrogens in an alkyl group may be optionally substituted with a hydroxy, cyano, thio, halo, nitro, amino, or alkoxy group.
  • the alkyl group is the side-chain of any naturally-occurring or modified L- or D-amino acid with an alkyl or substituted alkyl side-chain (e.g., alanine, beta-alanine, cyclohexylalanine, ethylglycine, valine, norleucine, leucine, norleucine isoleucine, allo-isoleucine, methionine, norvaline, homocysteine, cysteine, threonine, serine, homoserine, or 3-aminobutyric acid).
  • an alkyl or substituted alkyl side-chain e.g., alanine, beta-alanine, cyclohexylalanine, ethylglycine, valine, norleucine, leucine, norleucine isoleucine, allo-isoleucine, methionine, norvaline, homocysteine, cysteine, thre
  • Desirable aryl groups include monovalent aromatic hydrocarbon radicals consisting of one or more rings in which at least one ring is aromatic in nature, which may optionally be substituted with one of the following substituents: hydroxy, cyano, alkyl, alkoxy, thioalkyl, halo, haloalkyl, hydroxyalkyl, nitro, amino, alkylamino, diakylamino, or acyl.
  • Other suitable aryl groups include heteroaryl groups in which one or more carbons in a ring have been replaced with another atom, such as nitrogen, sulfur, or oxygen.
  • aryl groups include a phenyl, benzyl, or benzoyl moiety that is either unsubstituted or that contain one or more nitro, halo (e.g., chloro, fluoro, iodo, or bromo), aryl, (e.g., phenyl or benzyl), alkyl, alkoxy (e.g., methoxy), or acyl (e.g. acetyl or benzoyl) substituents.
  • a substituent is bound to carbon 2, 3, 4, 5, or 6 of one of these moieties.
  • Suitable aryl groups include benzoyl-NH-benzoyl and groups with fused aromatic rings, such as naphthalene, anthracene, phenanthrene, pyrene, and benzo[a]pyrene.
  • the aryl group is the side-chain of any naturally-occurring or modified L- or D-amino acid with an aryl or substituted aryl side-chain (e.g., phenylalanine, homophenylalanine, 2-napthylalanine, tyrosine, tryptophan, or tetrahydroisoquinoline-1-carboxylic acid).
  • Suitable alkoxy groups have the formula —OR, and suitable acyl groups have the formula —C(O)R, wherein R is an alkyl or aryl group as defined above.
  • alkoxy groups include, but are not limited to, methoxy, ethoxy, and isopropoxy groups.
  • acyl groups include acetyl and benzoyl groups.
  • acyl or guanylhydrozone substituents on the benzene rings in any of the structures shown above may be bound to any carbon in the ring, such as carbon 2, 3, 4, 5, or 6.
  • the benzene rings may be modified to contain other substituents, such as a nitro, halo, aryl, alkyl, alkoxy, or acyl substituent.
  • the compound is in a pharmaceutically acceptable carrier.
  • suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the composition can be adapted for the mode of administration and is typically in the form of a pill, tablet, capsule, spray, powder, or liquid.
  • the ARE-mRNA is an ARE-mRNA molecule identified by Bakheet et al. (supra), such as TNF ⁇ , an interleukin family member, an interferon family member, cyclooxygenase-1, or cyclooxygenase-2.
  • Other ARE-mRNAs are listed in FIGS. 4-7 .
  • the protein is encoded by any of the ARE-mRNAs identified by Bakheet et al. (supra) (FIGS. 4 - 7 ).
  • the condition that is treated or prevented in the clinical applications of the invention may include any condition associated with an ARE-mRNA or a protein encoded by an ARE-mRNA.
  • the condition is inflammation, fever, arthritis, septic shock, blood clot, stroke, acute respiratory distress syndrome, cachexia, or an autoimmune disease.
  • administration is oral or intravenous.
  • the subject is a mammal, for example, a human, cow, goat, horse, or mouse.
  • a compound of the invention is effective in at least 20, 40, 60, 80, or 90% of the subjects having a condition or an increased risk for a condition.
  • ⁇ ablates, reduces, or stabilizes symptoms in a subject suffering from the condition, prevents the onset of symptoms in a subject at risk for the condition, or results in a later age-at-onset of symptoms in the subject compared to the average age-of-onset for the corresponding untreated subjects.
  • ARE-mRNA an mRNA molecule containing an AU-rich element (ARE).
  • ARE AU-rich element
  • Many ARE-mRNAs have been identified and reported by Bakheet et al. (supra).
  • a list of ARE-mRNAs is available at the ARE Home Page (http://rc.kfshrc.edu.sa/ared/main.htm) which contains a database of human ARE-mRNAs. This database contains five clusters of ARE-mRNAs (http://rc.kfshrc.edu.sa/ared/diversity.htm, FIGS. 5 - 7 ). Additional ARE-mRNA molecules may be readily identified by one skilled in the art.
  • the method described by Bakheet et al. may be used to identify other mRNA molecules that contain the consensus motif WWWUAUUUAUWWW (SEQ ID NO: 1) with at most one mismatch in each direction of the nucleotides flanking the core UAUUUAU (SEQ ID NO: 2) pattern.
  • secretion of a protein encoded by an ARE-mRNA is meant the production of a protein encoded by an ARE-mRNA in a cell and the subsequent secretion of the protein from the cell. Protein production or secretion may be assayed in a number of ways, for example, using a reporter gene assay system, measuring the amount of a protein secreted from a cell, or using protein functional assays (see, for example, Humphreys et al. Cytokine 11:773-782, 1999).
  • a reporter gene assay that may be used to determine the effect of a candidate inhibitor on a step involved in secretion of a protein encoded by an ARE-mRNA is the luciferase assay described herein.
  • a standard Northern blot analysis may be used to measure the amount of ARE-mRNA produced in a cell
  • a standard ELISA or Western blot assay may be used to determine the amount of a protein encoded by an ARE-mRNA produced in a cell or secreted from a cell (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000).
  • inhibiting secretion of a protein encoded by an ARE-mRNA is meant decreasing the amount of a protein encoded by an ARE-mRNA that is secreted by a cell or tissue by administering a candidate inhibitor of secretion of a protein encoded by an ARE-mRNA.
  • the amount of TNF ⁇ secreted by a cell or tissue is compared to that secreted by a control cell or tissue that has not been administered the candidate inhibitor of secretion of a protein encoded by an ARE-mRNA or that has been administered a moderately active inhibitor of secretion of a protein encoded by an ARE-mRNA.
  • the amount of a protein encoded by an ARE-mRNA that is secreted by a cell or tissue after administration of a candidate inhibitor may be compared to that secreted by the cell or tissue before administration of the candidate inhibitor.
  • secretion of a protein encoded by an ARE-mRNA is decreased by at least 10, 30, 40, 50, 75, 90, or 95% relative to a control cell receiving vehicle only (for example, DMSO or saline).
  • a compound that inhibits secretion of a protein encoded by an ARE-mRNA reduces or stabilizes the level of ARE-mRNA or the encoded protein, the half-life of ARE-mRNA or the encoded protein, the processing of ARE-mRNA, the binding of ARE-mRNA or the encoded protein to a receptor or to another molecule, or the rate of passive or active diffusion of a protein encoded by an ARE-mRNA out of a cell, as measured using the cell-based assays described herein or in any other standard assay (see, for example, Ausubel et al., supra).
  • any of the cell-based assays may be performed in the presence of lipopolysaccharide (LPS) or any other activator of TNF ⁇ biosynthesis or secretion to increase the level of TNF ⁇ secretion, thereby facilitating the detection of a decrease in TNF ⁇ secretion caused by a candidate compound.
  • LPS lipopolysaccharide
  • the effect of a compound of the invention on the level of secretion of a protein encoded by an ARE-mRNA in a subject may be determined, if desired, by performing a routine ELISA assay on a serum sample from the subject to determine the amount of the protein that is present.
  • test compound or “candidate compound” is meant a naturally-occurring or artificially-derived chemical that is assayed for its ability to modulate secretion of a protein encoded by an ARE-mRNA or modulate ARE-mRNA regulation in one of the assay methods described herein or in any other appropriate assay.
  • treating is meant subjecting an animal, tissue, cell, cell lysate, cell extract, or molecule to a compound that modulates secretion of a protein encoded by an ARE-mRNA or modulates ARE-mRNA regulation.
  • condition is meant a state of being or feeling. Conditions include, but are not limited to, inflammation, inflammatory diseases, arthritis, autoimmune diseases, septic shock, blood clots, and injuries.
  • inflammation is meant an immune reaction that occurs when immunologically competent cells are activated in response to tissue damage, such as damage resulting from trauma, lack of blood supply, hemorrhage, foreign organisms, antigens, chemicals, irritants, allergens, electricity, heat, cold, microorganisms, surgical operations, or ionizing radiation.
  • tissue damage such as damage resulting from trauma, lack of blood supply, hemorrhage, foreign organisms, antigens, chemicals, irritants, allergens, electricity, heat, cold, microorganisms, surgical operations, or ionizing radiation.
  • antigens include viral, bacterial, protozoal, and fungal proteins, carbohydrates, and lipids.
  • Inflammation may be characterized by redness, swelling, heat, or pain, including hypersensitivity at the site of injury (primary hyperalgesia), hypersensitivity in neighboring non-injured tissue (secondary hyperalgesia), and diffuse pain.
  • any known animal model for inflammation may be used to test the ability of a compound of the present invention to prevent, stabilize, or treat inflammation.
  • Suitable models of inflammatory pain include unilateral injection of formalin, carrageenan, or complete Freund's adjuvant (CFA) into the hindpaw of rodent, such as a rat or mouse (Honor et al., J. Neruosci 19:7670-7678, 1999).
  • CFA complete Freund's adjuvant
  • mice or rats Honor et al., supra; Vieira et al., Eur. J. Pharmacol. 407:109-116, 2000).
  • amount sufficient to inhibit secretion of a protein encoded by an ARE-mRNA is meant an amount of a compound that decreases secretion of a protein encoded by an ARE-mRNA when administered to a subject.
  • the decrease in secretion of a protein encoded by an ARE-mRNA is at least 10%, 30%, 40%, 50%, 75%, or 90% greater in a treated subject than in an untreated, control subject.
  • the amount of the protein encoded by an ARE-mRNA that is secreted is at least 2, 5, 10, or 20-fold lower in a treated subject than in the same subject prior to the administration of the inhibitor or than in an untreated, control subject.
  • amount sufficient to modulate regulation of an ARE-mRNA is an amount of a compound that modulates regulation of an ARE-mRNA when administered to a subject.
  • the rate of mRNA degradation, mRNA processing, or mRNA translation in a treated subject is less than 90, 80, 70, 60, 50, 30, or 10% of the corresponding rate in an untreated, control subject.
  • the amount of an ARE-mRNA or a protein encoded by an ARE-mRNA is at least 2, 5, 10, or 20-fold lower in a treated subject than in the same subject prior to the administration of the inhibitor or than in an untreated, control subject.
  • regulation of an ARE-mRNA is measured by determining the decrease in the amount of the encoded protein that is effected by the administration of a compound to a subject.
  • a “derivative” is meant a structural derivative having a chemical modification that enhances the bioavailability, solubility, stability, or potency of a compound in vivo or in vitro or that reduces the toxicity of a compound in vivo or in vitro.
  • the inhibition of secretion of a protein encoded by an ARE-mRNA by the derivative is at least 10%, 30%, 40%, 50%, 75%, 90%, 95%, or 100% of that by the compound from which the derivative was derived.
  • modifications are known to those skilled in the field of medicinal chemistry.
  • condensing agent is meant a compound that accelerates the reaction between an amine and a carboxylic acid.
  • the condensing agent reacts with the carboxylic acid such that the hydroxyl group of the carboxylic acid is converted into a better leaving group for the nucleophilic substitution reaction between this activated carboxylic acid and the amine.
  • Condensing agents are well known in the art of organic synthesis.
  • FIG. 1 is a graph showing the effect of MES 10094 on luciferase activity.
  • FIG. 2 is a graph showing the effect of MES 10164 or MES 10170 on luciferase activity and TNF ⁇ secretion.
  • FIG. 3 is a graph showing the effect of MES 10171 or MES 10173 on luciferase activity.
  • FIG. 4 is a list of ARE-mRNAs using by Bakheet et al. (supra) in generating the ARE consensus motif (http://rc.kfshrc.edu.sa/ared/methodology.htm).
  • FIG. 5 is a list of ARE-mRNAs from the human ARE-mRNA database (Bakheet et al. (supra); http://rc.kfshrc.edu.sa/ared/diversity.htm). These ARE-mRNAs were clustered into five groups containing five, four, three, two, or one pentameric repeat within the ARE pattern (SEQ ID NOS: 3-7), respectively.
  • FIG. 5 lists the mRNAs in Clusters 1, 2, and 3. The mRNAs in Cluster 4 are listed in FIG. 6 , and the mRNAs in Cluster 5 are listed in FIG. 7 .
  • FIG. 6 is a list of ARE-mRNAs from Cluster 4 of the human ARE-mRNA database (Bakheet et al. (supra); http://rc.kfshrc.edu.sa/ARED/group4cluster.txt).
  • FIG. 7 is a list of ARE-mRNAs from Cluster 5 of the human ARE-mRNA database (Bakheet et al. (supra); http://rc.kfshrc.edu.sa/ARED/group5cluster.txt).
  • TNF ⁇ secretion a variety of compounds that inhibit TNF ⁇ secretion. These compounds are useful for clinical applications involving the treatment or prevention of diseases or injuries involving TNF ⁇ . Because of the high level of sequence identity between the ARE element in the 3′UTR of TNF ⁇ and the ARE element in other ARE-mRNAs, these compounds may also be used to inhibit secretion of other proteins encoded by ARE-mRNAs or to modulate the regulation of other ARE-mRNAs.
  • Some of the inhibitors of TNF ⁇ secretion that we identified are mono or bisguanylhydrazones having the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 2 and R 5 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • X is oxygen or H 2 N(HN)C(H)NN—, and m is at least one.
  • TNF ⁇ secretion is monoguanylhydrazones having the formula: or a derivative or salt thereof.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 2 and R 5 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group;
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl.
  • M is at least one.
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group; R 3 and R 4 are independently selected from the group consisting of an alkyl and an aryl group.
  • X is oxygen or H 2 N(HN)C(H)NN—.
  • R 3 or R 4 is a phenyl or methyl group.
  • R 1 or R 2 is a phenyl, 4-Cl-phenyl, 4-I-phenyl, 4-F-phenyl, 4-nitro-phenyl, 4-phenyl-phenyl, or 4-methoxy-phenyl group.
  • R 1 is a phenyl group and R 2 is an isopropyl group, or R 1 is an isopropyl group and R 2 is a phenyl group.
  • Additional inhibitory, monoguanylhydrazone compounds with a piperazine linker have the formula: or a derivative or salt thereof.
  • R 1 and R 4 are independently selected from the group consisting of an alkyl or an aryl group.
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • R 1 and R 5 are independently selected from the group consisting an aryl or alkyl group;
  • R 2 is hydrogen, an alkyl, or aryl group.
  • R 3 and R 4 are independently selected from the group consisting of hydrogen and an alkyl group.
  • X is oxygen or H 2 N(HN)C(H)NN—; and m is at least one.
  • R 1 and R 6 are independently selected from the group consisting of an alkyl and an aryl group;
  • R 3 is hydrogen, an alkyl, or an aryl group.
  • R 2 , R 4 , and R 5 are independently selected from the group consisting of hydrogen and an alkyl.
  • M is at least one.
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of hydrogen, an alkyl, and an aryl group.
  • M is at least one. In various embodiments, m is between 1 and 15, 1 and 10, or 1 and 5, inclusive.
  • TNF ⁇ secretion was confirmed in a standard ELISA assay that detected TNF ⁇ secreted by cells into the media (described further in Example 1). Additionally, the specificity of the compounds for inhibition of TNF ⁇ secretion was tested by comparing luciferase activity in cells expressing luciferase fused to the 3′UTR of TNF ⁇ to luciferase activity in control cells expressing luciferase without the heterologous 3′UTR of TNF ⁇ (Example 1).
  • a compound identified as capable of inhibiting secretion of a protein encoded by an ARE-mRNA or modulating regulation of an ARE-mRNA using any of the methods described herein may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.
  • the compound be optionally may administered as a pharmaceutically acceptable salt, such as a non-toxic acid addition salt or metal complex that are commonly used in the pharmaceutical industry.
  • acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
  • Metal complexes include zinc, iron, and the like.
  • the chemical compounds for use in such therapies may be produced and isolated as described herein or by any standard technique known to those in the field of medicinal chemistry.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the identified compound to patients suffering from a condition or at increased for a condition involving secreted TNF ⁇ . Administration may begin before or after the patient is symptomatic.
  • the therapy may be administered either directly to the site of inflammation (for example, by injection) or systemically (for example, by any conventional administration technique).
  • Administration of the compound may also be oral, topical parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmalic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, or intranasal.
  • the compound may be administered as part of a suppository.
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • the dosage of the therapeutic compounds in a pharmaceutically acceptable formulation depends on a number of factors, including the size and health of the individual patient. The dosage to deliver may be determined by one skilled in the art.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • treatment with a compound identified according to the methods described above may be combined with more traditional therapies for a disease characterized by inflammation, such as nonsteroidal anti-inflammatory drugs, or for a procoagulant condition, such as an anticoagulant.
  • Compounds that inhibit TNF ⁇ secretion may be purified or substantially purified, or may be one component of a mixture of compounds, such as a pool of chemical compounds.
  • inhibition of TNF ⁇ secretion is tested using progressively smaller subsets of the compound pool (e.g., produced by standard purification techniques such as HPLC or FPLC) until a single compound or minimal number of effective compounds is demonstrated to inhibit TNF ⁇ secretion.
  • a molecule that inhibits TNF ⁇ secretion is considered particularly useful as a therapeutic to decrease TNF ⁇ secretion in a patient with a condition in which deleterious TNF ⁇ secretion occurs, such as an inflammatory condition, arthritis, an autoimmune disease, fever, or stroke.
  • the candidate compounds were tested in a high-throughput, cell-based luciferase reporter assay.
  • a commercially available mouse macrophage Raw 264.7 cell line was stably transfected with a construct containing the CMV promoter operably linked to the luciferase gene which was modified using standard molecular biology techniques to contain the entire 3′ UTR of the TNF ⁇ gene. This 3′ UTR is important for the stability and translation of TNF ⁇ in cells.
  • a control cell line called “3B,” was generated by stably transfecting the 264.7 cell line with a commercially available construct containing the luciferase gene (without the addition of the heterologous 3′ UTR of the TNF ⁇ gene) under the regulation of the CMV promoter.
  • the effect of a candidate compound on luciferase activity in the 3B control cell line represents the global effect of the compound on any step required for expression or activity of luciferase protein, such as luciferase gene transcription, mRNA processing, mRNA stability, translation, protein stability, or enzymatic activity.
  • a candidate compound that decreases luciferase activity in the 5B cell line which contains the 3′ UTR of the qTNF ⁇ gene has little or no effect on luciferase activity in the control 3B cell line which does not contain the 3′ UTR of the TNF ⁇ gene.
  • the effect of the candidate compound is predominantly associated with TNF ⁇ rather than a global effect on any step required for expression or activity of a protein.
  • the luciferase activity in the 5B cell line treated with the DMSO vehicle control is similar to the luciferase activity in the control 3B cell line treated with the candidate compound.
  • the response of the 5B cell line (as a percent of the DMSO control) and the ratio of the luciferase activity in the 5B cell line (as a percent of the DMSO control) to the luciferase activity in the control 3B cell line (as a percent of the DMSO control) are similar.
  • the response of the 5B cell line and the 5B/3B ratio differ by less than 50, 40, 30, 20, 10, or 5%.
  • the above luciferase assay may be performed using the heterologous 3′UTR of an ARE-mRNA of interest instead of the 3′UTR of TNF ⁇ .
  • the polynucleotide sequences of many ARE-mRNAs are publicly available (see, for example, Bakheet et al., supra).
  • standard molecular biology techniques may be used to add the 3′UTR of an ARE-mRNA of interest to the luciferase gene, as described above.
  • TNF ⁇ secreted TNF ⁇ was also directly tested using an ELISA assay.
  • ELISA assay cells were incubated with 10 ⁇ M candidate compound for one hour, and then LPS was added. A standard ELISA analysis was used to measure the amount of TNF ⁇ secreted into the media (see, for example, Ausubel et al., supra). The effect of a candidate compound on TNF ⁇ secretion in this assay is reported in the following tables as a percentage of the amount of TNF ⁇ secreted by a control cell that was administered a DMSO vehicle control only.
  • the above ELISA assay may be performed using an antibody that is reactive with the protein of interest.
  • the cells may be treated with a compound that activates secretion of the protein of interest to facilitate the detection of candidate compounds that inhibit this secretion.
  • Chemical compounds that are found by the methods described above to effectively inhibit secretion of a protein encoded by an ARE-mRNA or to modulate regulation of ARE-mRNA in an in vitro or cell-based system may be tested further in animal models (such as those described in Examples 11 and 12). Particularly useful animal models include mouse and rat models of inflammation (see, for example, Kerland et al., Clin. Exp. Immunol. 115:32-41, 1999; and U.S. Pat. No. 6,022,900, incorporated herein by reference). Compounds that demonstrate an ability to decrease TNF ⁇ secretion in animal models may be used as therapeutics in humans to prevent, stabilize, or reduce TNF ⁇ secretion.
  • MES 10094 The ability of conformationally undefined guanylhydrazones to inhibit TNF ⁇ secretion was demonstrated by the inhibitory activity of MES 10094, shown below.
  • MES 10094 strongly inhibited luciferase activity in the 5B cell line and only weakly inhibited luciferase activity in the 3B cell line, producing a close correlation of the 5B and 5B/3B data (FIG. 1 ). This result suggests that MES 10094 effects expression or stability of the 3′ UTR of TNF ⁇ . Additionally, MES 10094 inhibited TNF ⁇ secretion after LPS stimulation. HPLC analysis of MES 11094 revealed that this compounds exists as only one bisguanylhydrazone isomer.
  • Luciferase Luciferase 5B/3B TNF ⁇ 5B (% of DMSO Secretion MES# (% of control 5B)/ (% of (10 DMSO (% of DMSO DMSO ⁇ M) GH R 1 Linker R 2 GH control) control 3B control) 11461 4Bn Phe P Leu 4Bn 28 31 44 11462 4Bn Phe P Leu 3Ac 24 37 55 11405 3Ac Phe P Leu 4Bn 39 49 55 11408 4Bn Leu P Leu 3Ac 59 54 11465 4Bn Phe P Ala 3Ac 72 71 11452 4Ac Phe P Leu 3Ac 13 50 88 11411 4Bn Ala Pr Leu 3Ac 65 71 11561 4Bn Phe Pr Leu 4Bn 24 26 41 11562 4Bn Phe Pr Leu 3Ac 44 38 50
  • R 1 and R 2 the amino acids listed (i.e., Phe, Leu, and Ala) represent the side chains that were used as the “R 1 ” or “R 2 ,” group illustrated in the general structure for initial library members shown above.
  • the “GH” column lists the type of guanylhydrazone present in the compound; for example, “4Bn” refers to a 4-benzoyl-benzamide derived guanylhydrazone moiety, and “3Ac” refers to a 3-acetyl-benzamide derived guanylhydrazone moiety.
  • “4-Bn Bn” denotes a 4-benzoyl-benzoyl group (i.e., C 6 H 5 —C(O)—C 6 H 4 —C(O)—).
  • “Ac” denotes an acetyl group (i.e., CH 3 C(O)—);
  • “Ph” denotes a phenyl group (i.e, C 6 H 5 ), and “Bzl” denotes a benzyl group (i.e, C 6 H 5 —CH 3 —).
  • MES 11461, 11462, 11405, 11408, 11465, 11411, 11561, and 11562 decrease TNF ⁇ secretion, with MES 11461, 11462, and 11561 producing the greatest inhibition.
  • Many of the compounds were further tested by assaying directly for TNF ⁇ polypeptide secretion.
  • MES 11461, 11462, 11405, 11452, 11561, and 11562 decreased the amount of secreted TNF ⁇ .
  • MES 11405 reversal of the interior component of MES 11462 (MES 11405) resulted in a molecule that displayed TNF ⁇ secretion inhibitory activity similar to that of MES 11461 and MES 11462.
  • MES 11461 HPLC analysis of MES 11461 indicated that the sample was a mixture of four bisguanylhydrazone isomers (MES 10164, 10165, 10166, and 10167, described further below) and probably four monoguanylhydrazone isomers, although only three fractions were observed (MES 10168, 10169, and 10170). These data are consistent with each 4-benzoylbenzamide derived guanylhydrazone existing in either an E or Z configuration relative to the central core. Similarly, MES 11462 was shown to comprise two bisguanylhydrazones (MES 10171 and 10172) and one monoguanylhydrazone (MES 10173).
  • each bisguanylhydrazone component of MES 11461 inhibited TNF ⁇ secretion, based on the luciferase reporter assay and direct measurement of TNF ⁇ secretion.
  • Both MES 10169 and 10170 inhibited luciferase activity in the 5B cell line and had minimal effect on the 3B cell line (FIG. 3 ).
  • each of the monoguanylhydrazone components of MES 11461 reduced the amount of secreted TNF ⁇ in the direct TNF ⁇ secretion assay.
  • a series of modifications of the MES library members were prepared. These modifications were divided into three groups: (i) amino acid analogs of the guanylhydrazone library members, (ii) a series of analogs of the monoguanylhydrazones, and (iii) a series of N-phenyl-4-piperazinyl succinate derivatives.
  • MES 10170 Z-mono isomer 1 Luciferase Luciferase 5B/3B TNF ⁇ 5B (% (% of DMSO Secretion of DMSO control 5R)/(% of (% of DMSO 10 ⁇ M GH R control) DMSO control 3B) control) MES Z 4-Bn Bn 33 39 41 10170 MES E/Z Bn 36 49 49 10222 MES B Bn 58 68 40 10249 MES Z Bn 37 49 40 10250 MES E/Z 4-NO 2 Bn 36 37 10224 MES E/Z 4-I Bn 23 26 10226 MES E/Z 4-Cl Bn 34 39 10227 MES E/Z 2-Naplithoyl 38 40 10228 ′′Bn′′ denotes benzoyl
  • MES 10170 Z-mono isomer 2 Luciferase Luciferase 5B/3B TNF ⁇ 5B (% (% of DMSO Secretion of DMSO control 5B)/(% (% of DMSO 10 ⁇ M GH R control) DMSO control 3B) of control) MES Z 4-Bn Bn 33 39 41 10170 MES E/Z Bn 34 33 49 10231 MES E Bn 37 41 39 10247 MES Z Bn 56 72 39 10248 MES E/Z 4-NO 2 Bn 36 40 42 10233 MES E/Z 3-pyr- 60 58 10234 CH 2 CH 2 CO MES E/Z C 4 H 3 N 2 CO 70 75 10235 ′′Pyr′′ denotes pyridinyl; ′′C 4 H 3 N 2 ′′ denotes pyrazine; and ′′Bn′′ denotes benzoyl.
  • guanylhydrazones were synthesized as described in Example 10 and assayed for TNF ⁇ secretion inhibitory activity (Table 11). These compounds have the general structure shown below in which X is either O or NN(H)C(NH)NH 2 .
  • NPPS N-phenyl-4-piperazinyl succinate
  • This synthetic method may be used to rapidly synthesize a variety of analogs by varying any of the R 1 -R 6 groups or by using other linker-containing compounds (compound 1).
  • a variety of naturally-occurring and modified amino acids (compounds 2 and 6) may be either obtained from commercial sources (such as Sigma and Novabiochem) as protected amino acids or protected using standard chemical methods.
  • any of the R 1 -R 6 groups may contain one of the suitable alkyl, aryl, or alkoxy groups described herein.
  • various benzoic acids (compounds 6 and 8) may be used in this method, such as those shown below.
  • R 1 -R 4 groups examples include alkyl groups, aryl groups, halogens, and —OR 5 groups in which R 5 is an alkyl group.
  • other linkers may be incorporated into these compounds by using other diamines with the nitrogen atoms separated by additional hydrocarbon moieties (e.g., 3, 4,5, 6, 7, 8, or more carbons) or with fewer hydrocarbon moieties (i.e., 1 carbon).
  • a piperazine linkage may be formed by using a piperazine containing compound, instead of the diamine compound 1 shown below.
  • one or more hydrogens in an amine group or hydrocarbon moiety of a linker may be substituted with a hydroxy, cyano, alkyl, aryl, alkoxy, thio, halo, nitro, or amino group.
  • one or more of the carbons in these linkers may be replaced with another atom, such as nitrogen, sulfur, or oxygen.
  • Other variations can also be introduced into the mono and bisguanylhydrazones through the incorporation of substituted aminoguanidine moieties, such as those shown below.
  • Suitable R 6 groups include any alkyl or aryl group.
  • n is an integer between 1 and 10, inclusive.
  • X is carbon, nitrogen, oxygen, or sulfur.
  • N-Methylmorpholine (20 mmol, 2.02 g, 2.20 mL) was added to the solution using a syringe, and the mixture was allowed to stir for 2-3 hours.
  • the reaction mixture was quenched by the addition of 30 mL of water to the reaction flask, and the resultant mixture was partitioned between ethyl acetate (600 mL) and water (600 mL).
  • the ethyl acetate layer was washed successively with saturated aqueous sodium bicarbonate (400 mL), water (4 ⁇ 600 mL), and brine (400 mL) and then dried over magnesium sulfate.
  • the catalyst was removed by filtration through a short pad of celite, and the celite was washed with minimal amounts of methanol (1-2 mL). The methanol in the solution was removed under vacuum on a rotary evaporator. To the remaining DMF solution was added the desired benzoic acid (compound 4) (2 mmol), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) (2 mmol, 886 mg), and N-methylmorpholine (3 equivalents, 6 mmol, 606 mg, 660 ⁇ L). The resulting solution was stirred overnight.
  • Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) (1.0 equivalents, 220 mg), the desired N-Boc-protected aminoacid (compound 6) (1.0 equivalent), and N-methylmorpholine (3.0 equivalents, 202 mg, 220 ⁇ L) were added, and the resulting mixture was stirred overnight.
  • the reaction mixture was poured into aqueous 0.2N NaOH (30 mL) and stirred for 30 minutes.
  • the resulting white solid was collected by filtration, washed with water (2 ⁇ 5 mL), and dried to leave the desired product.
  • Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) (1.0 equivalent, 44 mg), the desired benzoic acid (compound 8) (1.0 equivalent), and N-methylmorpholine (4.0 equivalents, 40 mg, 44 ⁇ L) were added, and the resulting mixture was stirred overnight.
  • the reaction mixture was poured into aqueous 0.2N NaOH (6 mL) and stirred for 30 minutes.
  • the resulting white solid was collected by filtration, washed with water (2 ⁇ 2 mL). and dried to leave the desired product.
  • N-(t-Butoxycarbonyl)ethylenediamine (compound 1) (10 mmol) was weighed directly into a clean dry flask, which was then tightly sealed with a stopper.
  • N-Cbz-(S)-Phenylalanine (compound 2) (10 mmol) was added directly to the amine in the flask. Under an inert atmosphere, dry dimethylformamide (30 mL) was added.
  • Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (10 mmol, 4.43 g) and then N-methylmorpholine (20 mmol, 2.02 g, 2.20 mL) were added to the solution, and the mixture was allowed to stir for 2-3 hours.
  • the reaction mixture was quenched by the addition of 30 mL of water to the reaction flask, and partitioned between water (600 mL) and ethyl acetate (600 mL.
  • the organic phase was washed successively with saturated sodium bicarbonate (400 mL), water (4 ⁇ 600 mL), and brine (400 mL) and then dried over magnesium sulfate.
  • Trifluoroacetic acid (1.2 equivalents, 2.4 mmol) was added to a solution of 1-[(S)—N-Cbz-2-Amino-3-phenylpropanoyl]-4-(t-butoxycarbonyl)-1,4-diazabutane (compound 3) (2mmol) in dimethylformamide (6 mL) and methanol (6 mL). Under a stream of nitrogen gas palladium on carbon (5 mol % of 10% on carbon, 106 mg) was carefully added. The reaction mixture was hydrogenated at 30 psi for ⁇ 30 minutes. Complete hydrogenation was assayed by thin layer chromatography to ensure that no starting material remains.
  • the catalyst was removed by filtration through a short pad of celite, and the celite was washed with minimal amounts of methanol (1-2 mL). The resultant solution was evaporated under reduced pressure to remove the methanol.
  • To the remaining DMF solution was added 4-acetylbenzoic acid (compound 4) (2 mmol), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (2 mmol, 886 mg) and N-methylmorpholine (3 equivalents, 6 mmol, 606 mg, 660 ⁇ L). The resulting solution was stirred overnight. The reaction was quenched by pouring into dilute aqueous sodium hydroxide (0.2 M, 120 mL). The resulting white solid was collected by filtration and washed with water (2 ⁇ 20 mL). The remaining solid was partially air dried under a stream of air, and fully dried under high vacuum to leave a white powdery solid.
  • MES 11461 was prepared in a manner analogous to the above examples employing N-(t-butoxycarbonyl)piperazine (as compound 1), N-Cbz-(S)-Phenylalanine (as compound 2), 4-benzoylbenzoic acid (as compound 4), N-(t-butoxycarbonyl)leucine (as compound 6), and 4-benzoylbenzoic acid (as compound 8).
  • the resulting crude guanylhydrazone product was a mixture of four bisguanylhydrazones and four monoguanylhydrazones which were separated by HPLC chromatography employing a reverse phase C 18 -capped silica gel column and an eluent consisting of 30% (0.5% trifluoroacetic acid in water) and 70% acetonitrile. Results from 1 H NMR analysis of three of these compounds is listed below.
  • MES 10164 (E,E)-Bisguanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(4-benzoylbenzoyl)-2-amino-4-methylpentanoyl]-1,4-piperazine bis trifluoroacetate.
  • MES 10165 (Z,Z)-Bisguanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(4-benzoylbenzoyl)-2-amino-4-methylpentanoyl]-1,4-piperazine bis trifluoroacetate.
  • MES 10168 (4-E)-Guanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(4-benzoylbenzoyl)-2-amino-4-methylpentanoyl]-1,4-piperazine trifluoroacetate.
  • MES 1142 was synthesized as described for the above examples. This particular example used N-(t-butoxycarbonyl)piperazine (as compound 1), N-Cbz-(S)-phenylalanine (as compound 2), 4-benzoylbenzoic acid phenylalanine (as compound 4), N-(t-butoxycarbonyl)leucine phenylalanine (as compound 6), and 3-acetylbenzoic acid phenylalanine (as compound 8).
  • the resulting crude guanylhydrazone product was a mixture of two bisguanylhydrazones and one monoguanylhydrazone which were separated by HPLC chromatography employing a reverse phase C 18 -capped silica gel column and an eluent consisting of 30% (0.5% trifluoroacetic acid in water) and 70% acetonitrile.
  • HPLC chromatography employing a reverse phase C 18 -capped silica gel column and an eluent consisting of 30% (0.5% trifluoroacetic acid in water) and 70% acetonitrile.
  • MES 10171 (E,E)-Bisguanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(3-acetylbenzoyl)-2-amino-4-methylpentanoyl]-1,4-piperazine bis trifluoroacetate.
  • MES 10172 (1-Z,4-E)-Bisguanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(3-acetylbenzoyl)-2-amino-4-methylpentanoyl]-piperazine bis trifluoroacetate.
  • MES 10173 (4-E)-Guanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(3-acetylbenzoyl)-2-amino-4-methylpentanoyl]-piperazine trifluoroaceteate.
  • MES 10249 and 10250 were prepared in a manner analogous to the above examples employing N-(t-butoxycarbonyl)piperazine (as compound 1), N-Cbz-(S)-phenylalanine (as compound 2), 4-benzoylbenzoic acid (as compound 4), N-(t-butoxycarbonyl)leucine (as compound 6), and benzoic acid (as compound 8).
  • the resulting crude guanylhydrazone product was a mixture of 2 monoguanylhydrazones which were separated by HPLC chromatography employing a reverse phase C 18 -capped silica gel column and an eluent consisting of 30% (0.5% trifluoroacetic acid in water) and 70% acetonitrile.
  • HPLC chromatography employing a reverse phase C 18 -capped silica gel column and an eluent consisting of 30% (0.5% trifluoroacetic acid in water) and 70% acetonitrile.
  • the 1 H NMR spectra are described below.
  • MES 10249 (E)-Guanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(benzoyl)-2-amino-4-methylpentanoyl]-1,4-piperazine trifluoroacetate.
  • MES 10250 (Z)-Guanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-[(2S)-N-(benzoyl)-2-amino-4-methylpentanoyl]-1,4-piperazine trifluoroacetate.
  • MES 10230 [1-[(S)-N-(4-benzoylbenzoyl)-2-amino-4-methylpentanoyl]-4[1-(4-(N-phenylpiperazine)succinyl]-1,4-piperazine]was prepared by using (S)-1-N-(4-Benzylbenzoyl)-2-amino-4-methylpentanoyl]-4-(t-butoxycarbonyl)-1,4-piperazine as the starting compound.
  • each of the R 1 -R 5 groups may be an alkyl, cycloalkyl, aryl, or heteroaryl group, such as those listed herein.
  • guanylhydrazones were prepared in a manner analogous to the above examples employing N-(t-butoxycarbonyl)ethylenediamine, N-Cbz-(S)-phenylalanine, 4-benzoylbenzoic acid, and 3-acetylbenzoic acid.
  • the resulting crude guanylhydrazone product was a mixture of two bisguanylhydrazones and one monoguanylhydrazone which were separated by HPLC chromatography employing a reverse phase C 18 -capped silica gel column and an eluent consisting of 30% (0.5% trifluoroacetic acid in water) and 70% acetonitrile.
  • MES 10092 (1-Z,4-E)-Bisguanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-(3-acetylbenzoyl)-1,4-diazabutane bis trifluoroacetate.
  • MES 10093 (4-E)-Guanylhydrazone of 1-[(S)-N-(4-benzoylbenzoyl)-2-amino-3-phenylpropanoyl]-4-(3-acetylbenzoyl)-1,4-diazabutane trifluoroacetate.
  • the compounds of the present invention can be assessed for their in vivo efficacy in treating inflammation.
  • a standard animal model of inflammation is utilized to directly test the efficacy of a candidate compound as an anti-inflammatory agent.
  • Paw swelling induced by injection of the irritant carrageenan into murine footpads is a standard technique used to detect clinically useful anti-inflammatory compounds.
  • C3H/HeN mice are divided into two groups.
  • One groups receives the vehicle of the candidate compound only through intraperitoneal injection, in each of its hindpaws (control 1), while the other group of mice receives the candidate compound and vehicle through intraperitoneal injection in each of its hindpaws.
  • Paw edema is induced by injection of 50 microliters of 1% Lambda-carrageenan in HEPES 25 mM, pH 7.4, into the planter surface of the left hindpaw of each mouse (test paw, receiving candidate compound and carraggeenan; and control 2, receiving candidate compound vehicle and carraggeenan), while the right paw is injected with 50 microliters of HEPES alone (control 3).
  • inflammation is measured by determining the thickness of each paw using a caliper.
  • a candidate compound that decreases carraggeenan-induced paw inflammation more than the vehicle control may be useful in clinical applications for the treatment or prevention of inflammatory or other TNF ⁇ -mediated conditions.
  • Candidate inhibitors of TNF ⁇ secretion may also be tested in vivo for their ability to prevent the lethal toxicity of lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • a candidate compound or vehicle control is administered to BALB/c mice and 1.5 hours later LPS is intraperitoneally injected into the mice in an amount sufficient to induce lethality due to endotoxic shock in 50% of the control mice within 72 hours.
  • LPS lipopolysaccharide
  • the ability of a candidate compound to decrease the frequency of death indicates that the compound decreases TNF ⁇ secretion in vivo.
  • Such compounds are useful in the treatment or prevention of fatal endotoxic shock and of other TNF ⁇ -mediated conditions.

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US9068234B2 (en) 2003-01-21 2015-06-30 Ptc Therapeutics, Inc. Methods and agents for screening for compounds capable of modulating gene expression
US20070111203A1 (en) * 2003-01-21 2007-05-17 Ptc Therapeutics Methods for identifying compounds that modulate untranslated region-dependent gene expression and methods of using same
US20050048549A1 (en) * 2003-01-21 2005-03-03 Liangxian Cao Methods and agents for screening for compounds capable of modulating gene expression
US9493845B2 (en) 2003-01-21 2016-11-15 Ptc Therapeutics, Inc. Methods for identifying compounds that modulate untranslated region-dependent gene expression and methods of using same
US9476870B2 (en) 2003-01-21 2016-10-25 Ptc Therapeutics, Inc. Methods and agents for screening for compounds capable of modulating gene expression
US8426194B2 (en) 2003-01-21 2013-04-23 Ptc Therapeutics, Inc. Methods and agents for screening for compounds capable of modulating VEGF expression
US8460864B2 (en) 2003-01-21 2013-06-11 Ptc Therapeutics, Inc. Methods for identifying compounds that modulate untranslated region-dependent gene expression and methods of using same
US20070072186A1 (en) * 2003-11-17 2007-03-29 Anuradha Mehta Methods and agents for screening for compounds capable of modulating her2 expression
US8217037B2 (en) 2006-04-07 2012-07-10 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Thiazole and thiophene analogues, and their use in treating autoimmune diseases and cancers
US8829014B2 (en) 2006-04-07 2014-09-09 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Thiazole and thiophene analogues, and their use in treating autoimmune diseases and cancers
US8741572B1 (en) 2007-06-20 2014-06-03 Ptc Therapeutics, Inc. Methods of screening for compounds for treating muscular dystrophy using mIGF1 mRNA translation regulation
US9376725B1 (en) 2007-06-20 2016-06-28 Ptc Therapeutics, Inc. Methods of screening for compounds for treating muscular dystrophy using human ALPHA7 integrin mRNA translation regulation
US8283115B1 (en) 2007-06-20 2012-10-09 Ptc Therapeutics, Inc. Methods of screening for compounds for treating muscular dystrophy using UTRN mRNA translation regulation
US9394539B1 (en) 2007-06-22 2016-07-19 Ptc Therapeutics, Inc. Survival motor neuron gene (SMN2) mRNA constructs for post-transcription regulation
US8283116B1 (en) 2007-06-22 2012-10-09 Ptc Therapeutics, Inc. Methods of screening for compounds for treating spinal muscular atrophy using SMN mRNA translation regulation

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